Tyrosine O-sulfation, a common post-translational modification of secretory and membrane proteins in higher eukaryotes, is mediated by a Golgi enzyme activity called tyrosylprotein sulfotransferase (TPST). TPST catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to tyrosines within acidic motifs of polypeptides. Several proteins involved in different aspects of vascular biology contain tyrosine sulfate, including adhesion molecules, coagulation factors, complement components, serpins, and extracellular matrix proteins. Tyrosine O-sulfation is known to be important for the biological function of P-selectin glycoprotein ligand-1, glycoprotein Ibalpha, coagulation factors V and VIII, complement factor C4, and others. The kinetics of the TPST reaction has been extensively studied using crude and partially purified enzyme preparations. However, the kinetic properties of the enzyme(s) in purified systems have not been explored, little is known about the regulation of TPST, and until recently nothing was known about the structure of TPST(s). We recently identified and cloned a family of two mammalian tyrosylprotein sulfotransferases, designated TPST-1 and TPST-2. The specific aims of this proposal are designed to compare the function of these two enzymes and to begin to address the broad question as to why more than one such enzyme exists. We hypothesize that TPST-1 and TPST-2 have distinct substrate specificities and may differentially modulate the biological function of a variety of proteins involved in diverse physiological functions. To explore this hypothesis the following specific aims are proposed. Aim 1 Determine the kinetic mechanism of TPST-1 and TPST-2. Aim 2 - Determine the substrate specificities of TPST-1 and TPST-2 toward various synthetic peptide and macromolecular substrates in vitro. Aim 3 - Determine the subcellular localization of TPST-1 and TPST-2. Aim 4. Define the pattern of TPST protein and mRNA expression in tissues and how TPST expression is regulated during inflammation and development. These studies should provide fundamental and new information on the function and regulation of TPSTs and provide insights into how the two known TPSTs might interact in biological systems.